Protein Profiling/Quantitative Proteomics
(Shixia Huang, Ph.D., Director)
The Dan L. Duncan Cancer Center is updating the services provided by "Protein Profiling/Quantitative Proteomics", as part of the existing Protein Expression/Proteomics Shared Resource led by Dr. Dean P. Edwards. These services are co-directed by Dr. Shixia Huang at Baylor College of Medicine and Dr. Eastwood Leung at Texas Children's Hospital and provides non-mass spectrometry (MS)-based platforms to measure the relative expression of multiple proteins in various biological samples. The following is a brief description of the services, technologies, prices and contact information.
Services at Baylor College of Medicine
Services at Texas Children's Hospital
Services at Baylor College of Medicine
Technology and Services:
Multiplex quantitative analyses using the suspension array system (Bio-plex from Bio-Rad) through Luminex xMAP bead technology.
Luminex’s xMAP technology is a microsphere-based multiplexing system. Multiplex assays provide attractive time and labor advantages over single-plex ELISA-based assays. In the Luminex assays, a specific antibody to the target protein is covalently coupled to internal fluorescently dyed beads. The beads with bound target proteins are separated by laser excitation and quantitated. Beads coupled with different antibodies, each with a distinct fluorescence signature, are mixed, thus enabling simultaneous assay of multiple protein targets in a single well of a 96-well plate. Assays can be done with small samples of cell and tissue extracts, cell culture media, serum, and other biologic fluids. Assays include:
- Cytokines, adipokines, & growth factors
- Intracellular signaling pathways
- Protein phosphorylation
- Many others (please inquire or check Luminex website for more information).
Depletion of IgG and albumin from serum samples to enrich low abundance proteins
In serum or plasma samples, the dynamic range of proteins spans over 10 orders of magnitude, much greater than the measurement capability of current technologies. Therefore, enrichment of the low abundance proteins becomes necessary for biomarker discovery or identification of protein differential expression in such samples. We have optimized the conditions for two different methods on depleting the high abundance proteins Albumin and IgG to produce large amount of enriched proteins for subsequent protein profiling analyses. We can also deplete other high abundance proteins in serum/plasma. The investigator provides serum/plasma samples, and we assay the protein concentration for each sample, delipidate the sample, deplete the most abundance proteins, assay protein concentration for depleted samples, and perform SDS-PAGE gel analysis for each sample.
Protein fractionation and protein profiling by two-dimensional liquid chromatograph (PF2D)
ProteomeLab PF2D (Beckman Coulter) is a high resolution 2-dimensional liquid protein fractionation system using chromatofocusing in the first dimension and a high capacity reverse phase HPLC column in the second phase. The system has an automated injection module for sample handling between first and second dimension columns, a sensitive UV detector and an automated 96-well format robot for collection of samples from the second dimension column. Selected liquid protein fractions can be further analyzed by mass spectrometry.
Antibody arrays for protein profiling
Antibody arrays enable identification of differentially expressed proteins between samples by applying fluorescently labeled proteins to a glass slide spotted with different antibodies. There are four different companies currently offering relatively large antibody arrays (500-725 antibodies on a single slide): ClonTech, Sigma, RayBiotech, and Full Moon BioSystems. Each array covers a different set of proteins with some overlapping. The pathways covered include apoptosis, cancer, cell cycle, nuclear receptors, cytokines and other extracellular regulatory pathways (see table below for details). The combination of 4 array platforms will enable us to assay more than 1500 unique proteins, the majority of which are among low abundance regulatory proteins important in cancer biology.
| Vendor | Functional groups | # of antibodies |
| ClonTech | Apoptosis, cancer, cell cycle, protein kinases, and neurobiology | 500 in duplicate |
| Sigma | Gene regulation, cell signaling, MAPK & PKC pathways, or p53 pathways | 725 |
| RayBiotech | Cytokines, adipokines, growth factors, chemikines, MMPS, phosphorylation, | 507 |
| Full Moon BioSystems | Intracellular Pathways: AKT, MAPK, NF-kB, JAK/STAT; nuclear and membrane receptors, tyrosine kinases, cancer/apoptosis, cell cycle, chromatin/transcription, cytoskeletal pathway, neuro-science, Insulin/Glucose | 656 in duplicate |
Protein-protein interaction arrays
We are in the process of testing different commercially available arrays for protein-protein interaction, including Invitrogen’s protoarray with >8000 proteins and Panomics’s protein arrays. We have obtained reproducible/reliable results with SH3 domain arrays. Each SH3 domain is a small, conserved sequence of about 60 amino acids that interacts with SH3 peptide ligands. This array platform is designed to identify the interaction partners with up to 152 SH3 domain containing proteins.
Data analysis
This is a R&D area for most of the platforms. Investigators are encouraged to analyze their own data or work with biostatisticians to analyze the data. Currently, the Core director is working with each investigator on the data analysis. We are also working with DLDCC Biostatistics and Data Management Shared Resource (Dr. Hilsenbeck and Dr. Chad Creigton) on antibody array analyses and Luminex data.
Luminex data analysis: a Bio-Plex Manager Software station is available for assessment of data quality, data analysis, and quantitation.
PF2D data analysis: ProteomeLab Software 32Karat is available for analysis of protein fractionations. For comparison between two or more samples, the newly released software package “Mapping Tool” (includes software ProteoVue, DeltaVue, and MultiVue) from Beckman Coulter is available.
Antibody array data analysis: GenePix Pro and Acuity software packages will be available for image analysis and data analysis.
Key instruments:
- Bio-plex 200 (Luminex 200) (Bio-rad)
- ProteomeLab PF2D system (Beckman Coulter)
- GenePix Axon Scanner 4200AL (Axon)
- SpectraMax 340PC 384 microplate reader (Molecular Devices)
2008-2009 Charge Back Fees
| Items | Price/sample |
| Luminex assays (highly customized) | Varies, inquire |
| Serum (50-100 ul serum) | $119 |
| Serum delipidation/depletion (up to 500 ul) | $222 |
| PF2D | $850*/$1008 |
| Antibody arrays | Varies, inquire |
| Array image analysis | $30/array |
* When supplies last (columns obtained with highly discounted price).
Location:
Margaret M. Alkek Building for Biomedical Research, Room R551
Personnel:
Shixia Huang, Ph.D., Director
Assistant Professor, Department of Molecular & Cellular Biology
Baylor College of Medicine
One Baylor Plaza
Margaret M. Alkek Building for Biomedical Research, Room R507
Houston, TX 77030
Phone: 713-798-8722
E-mail: shixiah@bcm.edu
Myra Custorio, B.S.,
Research Assistant, Department of Molecular & Cellular Biology
Baylor College of Medicine
One Baylor Plaza
Margaret M. Alkek Building for Medical Research, Room R551
Houston, TX 77030
Phone: 713-798-8935
E-mail: custorio@bcm.edu
Services at Texas Children's Hospital
The Genomics and Proteomics Core Laboratory (GPCL) of Texas Children's Hospital Cancer Centter operates as an integrated component under the network of the Baylor Dan L. Duncan Cancer Center. GPCL provides complementary protein profiling technology and subproteome purification services alongside the Protein Expression/Proteomics Shared Resource at Baylor. GPCL is directed by Dr. Eastwood Leung with the long term goal of biomarker discovery in complex biological samples. The laboratory provides two-dimensional gel electrophoresis-based protein profiling and subproteome purification such as glycoproteome or low abundance serum/plasma protein capturing. The following is a brief description of the services, technologies, prices and contact information.
Two-dimensional gel electrophoresis-based differential protein profiling
- Technology & instrumentation: Complex protein mixtures are separated by charge using isoelectric focusing in the first dimension. Further separation is achieved based on molecular mass of proteins using SDS-polyacrylamide gel electrophoresis in the second dimension. Over one thousand of protein spots can be resolved in optimized condition. Two gel formats are available: 1) The Zoom®IPGRunner? together with Novex gel from Invitrogen Inc consititute the minigel (8cm) format; 2) IPGphor II for the first dimension and Ettan DALTsix system for the second dimension from GE Healthcare Inc. constitute the large gel format. Protein gels can be stained with mass spectrometry compatible silver stain, sypro ruby, or Commassie blue stain. 2-D fluorescence differnece gel electrophoresis (DIGE) analysis will be available soon. Differential protein spots can be excised and processed for subsequent mass spectrometry analysis.
Subproteome purification
- Glycoproteome purification: Hydrazine-based solid phase extraction of glycoproteome is available. Depletion of abundance proteins in plasma or serum can be employed to enhance the discovery of low abunbance protein markers in body fluid. Captured subproteome will be subjected to gel electrophoresis-based separation.
Location: Feigin Center (1102 Bates Street, Houston, TX77030) Room C1070.01
Director:
Eastwood Leung Ph.D.
Assistant Professor
Dan L. Duncan Cancer Center
Dept. of Pediatrics and Dept of Cellular and Molecular Biology
Phone: 832-824-4373
E-mail: exleung@texaschildrenshospitals.org
Personnel:
Matthew Folsom B.A., CLDir, CLSp (CG)
Laboratory manager
Office: 832-824-4458
Email: mrfolsom@texaschildrenshospital.org
Sergery Torsky, Ph.D.
Bioinformatics programmer
Phone: 832-824-1036
Sivashankarappa Gurusiddappa
Staff Technologist
Phone: 832-824-4453
Main Laboratory:
Phone: 832-824-4354
Fax: 832-825-4038
Prices
| Services | Price/Sample |
|---|---|
| Isoelectric focusing small gel format | $25 |
| Isoelectric focusing large gel format | $30 |
| 2nd dimension SDS-PAGE small gel format | $40 |
| 2nd dimension SDS-PAGE large gel format | $60 |
| Commassie blue stain of small gel | $10 |
| Commassie blue stain of large gel | $30 |
| Sypro ruby stain of small gel | $30 |
| Sypro ruby stain of large gel | $80 |
| Mass spectrometry-compatible silver stain of small gel | $25 |
| Mass spectrometry-compatible silver stain of large gel | $55 |
| 2D gel image analysis | $25 |
| Hydrazine-based glycoprotein capture assay | $75 |
More information of the GPCL in the Texas Children's Hospital